An important objective in the post-genome area is the detect-ion of trace amounts of expressed protein/peptide expressed through genes and the separation and identification thereof. In the past, peptide fingerprinting following two-dimensional electrophoresis was commonly used to achieve this objective (see Non-patent Document 1). However, this method had problems with reproducibility of the method due to the complex procedure Separation and identification methods using multi-dimensional high-performance liquid chromatography (multi-dimensional HPLC), and techniques using ICAT have recently been proposed to overcome this problem (see Non-patent Document 2).
Among these methods, methods for separating and identifying protein/peptide directly by multi-dimensional HPLC have the shortcoming of requiring considerable labor and time since all proteins/peptides are processed simultaneously. In addition, methods using ICAT attempt to comprehensively analyze protein/peptide by labeling the thiol groups of thiol-containing protein/peptide with an isotope-coded affinity tag (ICAT) reagent, capturing the protein/peptide with a biotin-coupled column, subjecting all of the proteins/peptides to enzymatic hydrolysis, separating the resulting mixture of peptide fragments by HPLC, and carrying out mass spectrometry on the peptide fragments with a mass spectrometer (MS). However, since this method involves subjecting all thiol-containing protein/peptide to enzymatic hydrolysis, it has the shortcoming of fragments of non-target protein/peptide present in large amounts impairing detection and identification of target trace protein/peptide, thereby creating the need to achieve further improvement in this technical field.    [Non-Patent Reference 1] Dunn M J. Two-dimensional gel electrophoresis of proteins, J Chromatogr 1987; 418:145-185
[Non-Patent Reference 2] Gygi S. P, Rist B, Gerber S. A, Turecek F. Gelb M. H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, Nature Biotechnology 1999; 17:994-999